Diagnostic test for equine arteritis virus mediated disease

ABSTRACT

This invention relates to recombinant DNA and polypeptides encoded thereby which have use in the provision of vaccines, diagnostic test kits and methods of diagnosis and treatment or prophylaxis for equine arteritis virus (EAV) and EAV mediated disease.

This application is a 371 of PCT/GB76/01505 filed Jun. 20, 1996.

The present invention relates to recombinant DNA and polypeptides encoded thereby, having use in provision of antibodies, vaccines, diagnostic test kits and methods of diagnosis and treatment or prophylaxis for equine arteritis virus (EAV) and equine arteritis virus mediated disease.

Equine viral arteritis, a disease for which equids are the only reported hosts, has been known for some 40 years and manifests itself with widely varying clinical signs. In its most severe form EAV infection causes abortion and foal death which makes it a potentially significant commercial threat to, inter alia, the horse breeding industry. Early veterinary articles refer to it as epizootic cellulitis, pinkeye or equine influenza. Disease outbreaks are identified infrequently and field isolates of the single stranded RNA virus itself are rare.

EAV is transmitted by the respiratory and venereal routes, with a 30-60% carrier state existing in seropositive stallions which persistently shed virus in their semen. Thus the venereal route is a particular cause for concern, as these shedding stallions may infect broodmares at mating. In the light of the potential economic importance of the virus and its stud carrier mediated infection capability there exists a requirement for prophylactic treatment and reliable diagnosis of equine viral arteritis, and rapid identification of equids previously exposed to the infectious agent. Laboratory tests based on ELISA using whole virus as antigen, virus neutralisation (VN) and complement fixation (CF) formats have been developed (see Chirnside (1992) Br Vet J 148, pp 181). The known ELISA is relatively sensitive when applied to tissues, eg sera, from horses previously vaccinated for other diseases such as influenza and herpesvirus, while the VN and CF formats have limited temporal sensitivity; the VN test is unable to distinguish between vaccination and natural infection.

Vaccination procedures have concentrated on safety and efficacy of whole inactivated virus and attenuated live virus vaccines. The live vaccine can induce short term shedding of virus from the nasopharynx and does not prevent this causing infection of commonly housed animals which have not been so treated. It is not yet known if formalinised vaccines or other inactivated vaccine preparations provide reliable protection.

Attempts to provide improvements to both diagnostic tests and vaccines have included studies into panels of antibodies raised against various EAV proteins. A 29K envelope protein (G_(L)) has been identified as antigenic (Balasuriya et al, (1993) J Gen Virol 74, 2525-2529; Deregt et al, (1994) J Gen Virol 75, 2439-2444) and peptides derived from this protein induce a virus neutralising response in horses (Chirnside et al, (1995) J Gen Virol in press). A previous invention (UK Patent Application No GB 9400656.4) provided isolated peptides of G_(L) that produce a potent neutralising immune response against EAV when administered to animals, particularly horses, and these peptides provided sensitive detection of EAV antibodies when used as binding agents in biding assays formats. Further provided was DNA encoding for these peptides.

In the first aspect of the present invention there is provided a method for testing for the presence of antibodies to equine arteritis virus comprising use of a peptide or peptide conjugate of the viral nucleocapsid (N) protein as a specific binding agent. Such test is preferably of ELISA format but may use the peptide or conjugate as immobilised binding agent or labelled secondary binding agent in a so-called sandwich assay. Preferred peptides or peptide conjugates of the invention comprise epitopes present in the amino acid sequence corresponding to amino acids 1 to 110 (SEQ ID No.2) of EAV N, more preferably amino acids 1 to 69 of EAV N (SEQ ID No.3) and, additionally, amino acids 1 to 28 (SEQ ID No.4), or a sequence having at least 90% homology to these sequences. These peptides are antigenic and may be used to provide isolated antibodies produced by an immunological response when a suitable host is exposed to the antigen.

In a binding assay where the peptide or peptide conjugate is immobilised this method may conveniently be carried out by use of commercially available assay plates onto which the peptide or conjugate is coated by suitable incubation in the known manner. For the purpose of assay, a sample to be screened for EAV antibodies, eg a serum sample, is typically incubated in contact with the plate, eg in the wells, whereafter any EAV antibody present is identified by exposure to eg an anti-horse IgA, IgM, IgG or other immunoglobulin conjugated to a reporter group. Such reporter group may be in the form of radiolabel, chemical label or a biological label. A typical biological label is an enzyme or cofactor, eg biocin, and is detected by exposure to all the reactants necessary for a reporter reaction to occur dependent upon the presence of the reporter group. In the case of biotin the well may be exposed to screptavidin-peroxidase and the substrate o-phenylenediamine dihydrochloride and the absorbance of the plate determined at 490 nm.

In a further example, an immobilised anti-horse IgA, IgM, IgG or ocher immunoglobulin antibody raised in another animal, may be used to bind a specific class of horse antibody; the immobilised horse antibody provided may then be exposed to a solution containing labelled peptide or conjugate of the invention whereby the presence of anti-EAV antibody is indicated by assay of the amount of label present. Other assay formats such as competitive assays using either bound or unbound peptide or conjugate will occur to those skilled in the art; these will include simple observation of agglutination between peptide or conjugate and the antibody in a simple dilution test.

In a further aspect of the present invention there are provided test kits for use in carrying out the assay of the invention characterised in that they comprise a peptide, peptide-conjugate or antibodies of the invention, together with optional agents and items necessary for performing such assays. Such agents and items may include other binding agents or color forming agents such as labelled antibodies eg biotinylated anti-horse IgG, horseradish peroxidase, streptavidin peroxidase conjugate and o-phenylenediamine dihydrochloride. It will be realised that the term peptide and peptide conjugate as used herein will encompass oligopeptides, polypeptides and proteins as long as they fulfill the criteria of the invention with regard to the immunological activity and content of epitopic sequences. The term conjugate designates conjugation to any physiologically acceptable entity.

The peptides, peptide conjugates and binding assays of the present invention will now be described, by way of example only, by reference to the following sequence listing, figures and examples.

SEQUENCE LISTING

SEQ ID NO: 1: is the DNA sequence equivalent to the entire EAV genome minus the first 18 bases and the polyA tail.

SEQ ID NO: 2: is the amino acid sequence of the virus nucleocapsid protein (N) encoded by open reading frame (ORF) 7, and that is fused in-frame to GST in FP70 to express rN1-110.

SEQ ID NO: 3: is the amino acid sequence corresponding to amino acids 1 to 69 of the EAV N protein, and that is fused in-frame to GST in FP71 to empress rN1-69.

SEQ ID NO: 4.: is the amino acid sequence corresponding to amino acids 1 to 28 of the EAV N protein, and that is fused in-frame to GST in FP7Fspl to express rN1-28.

FIGURES

FIG. 1 shows an immunoblot of purified fusion proteins (Fps 70, 71, 72, 73 and 7Fspl) and glutathione-S-cransferase (Gst) with serum grom an individual horse pre- and post-EAV infection. For plasmid and fusion protein derivations see Table 1.

FIG. 2 shows equine sera ELISA absorbance values to recombinant EAV N proteins. ELISA plates were coated with 0.5 μg per well of purified fusion protein (FP) or glutathione-S-transferase (GST). Sera were tested in two replicate wells to each antigen and the absorbance of each well read at 490 nm. The GST absorbance was subtracted from the FP absorbance to derive an EAV-specific value. Each bar represents the mean value from two replicates of each serum. Cut-off points determining ELISA seropositivity for each antigen, calculated from the absorbance values of 8 VN- control sera are shown as a horizontal line on each graph: FP70=0.592; FP71=0.483; FP72=0.294; FP73=0.407; FP7Fsp1=0.582. The virus neutralising titre (VN titre) of the 8 sera tested are shown on the x-axis as log₁₀ VN titres.

EXAMPLE 1

Production of peptides and conjugates of the invention and DNA and vectors encoding therefore.

cDNA encompassing EAV open reading frame (ORF) 7 (den Boon et al [1991], J Virol, 65, 2910-2920; de Vries et al, [1992], J Virol 66, 6294-6303) corresponding to the EAV N protein was cloned into the bacterial vectors pGEX-3X and pGEX-2T (Table 1) and constructs screened for fusion protein expression using PAGE with cloning confirmed by RE digestion analysis and sequencing over the plasmid/insert junctions. Plasmids are referred to as FPx, the expressed recombinant fusion proteins as rNy-z (where y and z refer to amino acid residue numbers in EAV N). Affinity purified glutathione-S-transferase (GST) fusion proteins were screened for reactivity in immunoblots with a panel of pre- and post-EAV-infection equine sera. Although horse sera exhibit some background absorbence to GST in immunoblots, post-infection sera bound strongly and specifically to fusion proteins containing amino acids N1-110 and N1-69, and failed to bind fusion proteins containing NI-28, N70-89 and N90-110 specifically (FIG. 1).

EXAMPLE 2

ELISA using EAV nucleocapsid (N) fusion proteins

Dynatech Immulon 3 microtitre plates were coated with rN1-69 or rN1-28 antigen by exposure to 100 μl of 5μg/ml antigen in 0.05 M carbonate buffer at pH9.6 (Sigma cat No C3041) at 4° C. overnight.

Plates were washed three times with phosphate buffered saline (PBS) containing 0.05% Tween 3 20 (PBST) and then blocked with 100l PBST containing 5% normal goat serum (Seralab) (PBSTG) for 1 hour at 37° C. Plates were washed again three times with PBST to render them ready for use.

Test sera were diluted 1:100 in PBSTG and 100 μl of this solution added to wells prepared as above and incubated for 90 minutes at 37° C. Plates were washed again three times with PBST and solution prepared by diluting goat anti-horse IgG biotin conjugate (KPL catalogue No 162102) 1:1000 in PBSTG and adding 1001 to each well before being incubated for 90 minutes at 37° C. Plates were washed three times with PEST and a solution prepared by diluting streptavidin-peroxidase conjugate (KPL catalogue No 143000) 1:1000 in PBSTG and adding 100 μl to each well before incubating at room temperature for 30 minutes. Plates were washed three times with PBST and 100 μl O-phenylenediamine dihydrochloride (Sigma cat. No P8287) (0.5 mg/ml in 0.05 phosphate citrate buffer, pH5.0, Sigma cat. No. P4922)) added to each well and incubated for 10 minutes at room temperature. 50 μl 4M H₂ SO₄ was added to stop the reaction and absorbence read at 490 nm. Since horse sera at a 1:100 dilution can bind native GST it is necessary to subtract absorbence readings obtained for sera against GST from the GST-fusion protein absorbence. Each serum is tested in duplicate wells against each is antigen.

FIG. 2 shows the results of 8 VN equine sera in ELISA to different recombinant EAV N proteins. Cut-off points determining ELISA seropositivity for each antigen, calculated from the value of 8VN negative equine sera, are shown as a horizontal line on each graph (rN1-110=0.592; rN1-69=0.483; rN70-89=0.294; rN90-110=0.407; rN1-28=0.582). From these results rN1-69 and rN1-28 were identified as suitable antigens for the detection of EAV-specific antibodies in ELISA.

EXAMPLE 3

ELISA using rN1-69 and rN1-28 binding agents

Panels containing seronegative and virus neutralising sera were tested in ELISA to purified rN1-69 and rN1-28 (Table 2). In ELISAs a recombinant fusion protein containing residues 1-69 or 1-28 discriminated between pre- and post-infection equine sera. In additional ELISA tests screening pre- and post-EAV vaccination samples and including isolate specific sera, the rNI-69 and rN1-28 antigens were able to discriminate between samples pre- and post vaccination with Artervac (commercial inactivated virus vaccine), even in the absence of vaccination induced neutralising antibody, and detect isolate-specific VN sera as seropositive in ELISA. The mean absorbence rising following vaccination were 1.240+0.690 and 0.495+0.352 for rN1-69 and rN1-28 respectively.

                                      TABLE 1                                      __________________________________________________________________________     Nucleocapsid gene constructs and fusion proteins                                   Amino acid                                                                           Fusion                                                                             Fusion                                                           Plasmid                                                                            residue                                                                              protein                                                                            protein size                                                                         EAV   Restriction                                                                            pGEX vector                                  (FP)                                                                               (N)   (rN)                                                                               (kDa) cDNA clone                                                                           digest  restriction digest                           __________________________________________________________________________     70  -3.sup.1 -110                                                                        1-110                                                                              42    106.sup.2                                                                            HindIII (12305) -                                                                      3X x HindIII                                                           HindIII.sup.v (>12700)                               71  -3.sup.1 -69                                                                         1-69                                                                               36    FP70  HindIII (12305) -                                                                      3X x BamhI                                                             RsaI (12523)                                                                           EcoRI.sup.κ                            72  70-89 70-89                                                                              30    FP70  RsaI (12524) -                                                                         2T x SmaI                                                              RsaI (12583)                                         73  90-10 90-110                                                                             30    FP70  RsaI (12584) -                                                                         2T x SmaI                                                              EcoRI.sup.v (.12700)                                 7Fsp1                                                                              -3.sup.1 -28                                                                         1-28                                                                               31    106.sup.2                                                                            HindIII.sup.k (12305) -                                                                3X x SmaI                                                              FspI (12399)                                         __________________________________________________________________________      .sup.κ 3' recessed end filled in with the Klenow fragment of DNA         polymerase                                                                     .sup.v Vector derived                                                          .sup.1 The negative number corresponds to additional amino acids cloned        into pGEX which are not encoded by ORF 7                                       .sup.2 see de Vries et al 1990, Nuclei Acids Research 18, 3241-3247.     

                  TABLE 2                                                          ______________________________________                                         Comparison of virus neutralising antibody                                      titres and ELISA absorbence values                                                     Log.sub.10 VN                                                                           VN      rN1-69                                                                               rN1-69                                                                               rN1-28                                                                               rN1-28                                      antibody test    ELISA ELISA ELISA ELISA                               Equine sera                                                                            titre.sup.1                                                                             result  A.sub.490                                                                            result.sup.b                                                                         A.sub.490                                                                            result.sup.c                        ______________________________________                                         Negative controls                                                              32277   0        -       0.126 -     0.170 -                                   32278   0        -       0.156 -     0.310 +                                   32779   0        -       0.098 -     0.180 -                                   32280   0        -       0.095 -     0.101 -                                   32281   0        -       0.115 -     0.155 -                                   32282   0        -       0.115 -     0.134 -                                   32283   0        -       0.161 -     0.204 -                                   32284   0        -       0.152 -     0.222 -                                   Post infection                                                                 32252   3.6      -       3.746 +     2.250 +                                   32255   2.475    +       2.504 -     0.679 +                                   32257   2.625    +       3.660 +     1.856 +                                   32258   2.700    +       3.520 +     1.182 +                                   32259   2.550    +       2.536 +     0.650 +                                   32260   2.850    +       1.238 -     0.314 +                                   32261   1.875    +       2.753 +     2.024 +                                   32262   2.475    +       3.00  +     0.920 +                                   Paired vaccination samples                                                     33745 pre                                                                              0        -       0.132 -     0.230 -                                       post                                                                               0.3      -       0.664 +     0.340 +                                   33746 pre                                                                              0        -       0.353 +     0.344 +                                        post                                                                              0.45     -       0.967 +     0.580 +                                   33747 pre                                                                              0        -       0.168 -     0.256 -                                        post                                                                              0.525    -       2.427 +     1.212 +                                   33962 pre                                                                              0        -       0.157 -     0.170 -                                        post                                                                              0        -       0.884 +     0.387 +                                   33963 pre                                                                              0        -       0.117 -     0.145 -                                        post                                                                              0.9      +       2.144 +     0.997 +                                   33964 pre                                                                              0        -       0.156 -     0.175 -                                       post                                                                               0        -       1.572 +     0.851 +                                   33435 pre                                                                              0        -       0.348 +     0.286 -                                       post                                                                               0.3      -       1.452 +     0.491 +                                   35097 post                                                                             1.5      +       3.226 +     2.026 +                                   35098 post                                                                             1.5      +       3.441 +     1.908 +                                   Isolate specific                                                               Bucyrus 3.1      -       3.249 +     1.110 +                                   84-KY-Al                                                                               2.5      -       0.888 -     0.288 -                                   Wroclaw-2                                                                              2.2      +       0.424 +     0.276 -                                   Arvac   2.5      -       0.422 +     0.319 +                                   Killed  1.9      -       1.117 +     0.620 +                                   Bucyrus                                                                        ______________________________________                                          .sup.a Log.sub.10 VN antibody titre ≧ 0.6 is deemed seropositive i      the EAV VN neutralising test                                                   .sup.b the cut off value to determine seropositive status was taken as         (mean +2SD) of the 8 VN negative control sera (positive ≧ 0.177)        .sup.c the cut off value to determine seropositive status was taken as         (mean + 2SD) of the 8 VN negative control sera (positive ≧ 0.308)       pre = prevaccination serum sample                                              post = postvaccination serum sample                                      

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES: 4                                              - (2) INFORMATION FOR SEQ ID NO: 1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #pairs    (A) LENGTH: 12687 base                                                         (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: cDNA                                                 #ID NO: 1:(xi) SEQUENCE DESCRIPTION: SEQ                                       - TGCCATATAC GGCTCACCAC CATATACACT GCAAGAATTA CTATTCTTGT GG - #GCCCCTCT          60                                                                           - CGGTAAATCC TAGAGGGCTT TCCTCTCGTT ATTGCGAGAT TCGTCGTTAG AT - #AACGGCAA         120                                                                           - GTTCCCTTTC TTACTATCCT ATTTTCATCT TGTGGCTTGA CGGGTCACTG CC - #ATCGTCGT         180                                                                           - CGATCTCTAT CAACTACCCT TGCGACTATG GCAACCTTCT CCGCTACTGG AT - #TTGGAGGG         240                                                                           - AGTTTTGTTA GGGACTGGTC CCTGGACTTA CCCGACGCTT GTGAGCATGG CG - #CGGGATTG         300                                                                           - TGCTGCGAAG TGGACGGCTC CACCTTATGC GCCGAGTGTT TTCGCGGTTG CG - #AAGGAATG         360                                                                           - GAGCAATGTC CTGGCTTGTT CATGGGACTG TTAAAACTGG CTTCGCCAGT TC - #CAGTGGGA         420                                                                           - CATAAGTTCC TGATTGGTTG GTATCGAGCT GCCAAAGTCA CCGGGCGTTA CA - #ATTTCCTT         480                                                                           - GAGCTGTTGC AACACCCTGC TTTCGCCCAG CTGCGTGTGG TTGATGCTAG GT - #TAGCCATT         540                                                                           - GAAGAGGCAA GTGTGTTTAT TTCCACTGAC CACGCGTCTG CTAAGCGTTT CC - #CTGGCGCT         600                                                                           - AGATTTGCGC TGACACCGGT GTATGCTAAC GCTTGGGTTG TGAGCCCGGC TG - #CTAACAGT         660                                                                           - TTGATAGTGA CCACTGACCA GGAACAAGAT GGGTTCTGCT GGTTAAAACT TT - #TGCCACCT         720                                                                           - GACCGCCGTG AGGCTGGTTT GCGGTTGTAT TACAACCATT ACCGCGAACA AA - #GGACCGGG         780                                                                           - TGGCTGTCTA AAACAGGACT TCGCTTATGG CTTGGAGACC TGGGTTTGGG CA - #TCAATGCG         840                                                                           - AGCTCTGGAG GGCTGAAATT CCACATTATG AGGGGTTCGC CTCAGCGAGC TT - #GGCATATC         900                                                                           - ACAACACGCA GCTGCAAGCT GAAGAGCTAC TACGTTTGTG ACATCTCTGA AG - #CAGACTGG         960                                                                           - TCCTGTTTGC CTGCTGGCAA CTACGGCGGC TACAATCCAC CAGGGGACGG AG - #CTTGCGGT        1020                                                                           - TACAGGTGCT TGGCCTTCAT GAATGGCGCC ACTGTTGTGT CGGCTGGTTG CA - #GTTCTGAC        1080                                                                           - TTGTGGTGTG ATGATGAGTT GGCTTATCGA GTCTTTCAAT TGTCACCCAC GT - #TCACGGTT        1140                                                                           - ACCATCCCAG GTGGGCGAGT TTGTCCGAAT GCCAAGTACG CAATGATTTG TG - #ACAAGCAG        1200                                                                           - CACTGGCGCG TCAAACGTGC AAAGGGCGTC GGCCTGTGTC TCGATGAAAG CT - #GTTTCAGG        1260                                                                           - GGCATCTGCA ATTGCCAACG CATGAGTGGA CCACCACCTG CACCCGTGTC AG - #CCGCCGTG        1320                                                                           - TTAGATCACA TACTGGAGGC GGCGACGTTT GGCAACGTTC GCGTGGTTAC AC - #CTGAAGGG        1380                                                                           - CAGCCACGCC CCGTACCAGC GCCGCGAGTT CGTCCCAGCG CCAACTCTTC TG - #GAGATGTC        1440                                                                           - AAAGATCCGG CGCCCGTTCC GCCAGTACCA AAACCAAGGA CCAAGCTTGC CA - #CACCGAAC        1500                                                                           - CCAACTCAGG CGCCCATCCC AGCACCGCGC ACGCGACTTC AAGGGGCCTC AA - #CACAGGAG        1560                                                                           - CCACTGGCGA GTGCAGGAGT TGCTTCTGAC TCGGCACCTA AATGGCGTGT GG - #CCAAAACT        1620                                                                           - GTGTACAGCT CCGCGGAGCG CTTTCGGACC GAACTGGTAC AACGTGCTCG GT - #CCGTTGGG        1680                                                                           - GACGTTCTTG TTCAAGCGCT ACCGCTCAAA ACCCCAGCAG TGCAGCGGTA TA - #CCATGACT        1740                                                                           - CTGAAGATGA TGCGTTCACG CTTCAGTTGG CACTGCGACG TGTGGTACCC TT - #TGGCTGTA        1800                                                                           - ATCGCTTGTT TGCTCCCTAT ATGGCCATCT CTTGCTTTGC TCCTTAGCTT TG - #CCATTGGG        1860                                                                           - TTGATACCCA GTGTGGGCAA TAATGTTGTT CTGACAGCGC TTCTGGTTTC AT - #CAGCTAAT        1920                                                                           - TATGTTGCGT CAATGGACCA TCAATGTGAA GGTGCGGCTT GCTTAGCCTT GC - #TGGAAGAA        1980                                                                           - GAACACTATT ATAGAGCGGT CCGTTGGCGC CCGATTACAG GCGCGCTGTC GC - #TTGTGCTC        2040                                                                           - AATTTACTGG GGCAGGTAGG CTATGTAGCT CGTTCCACCT TTGATGCAGC TT - #ATGTTCCT        2100                                                                           - TGCACTGTGT TCGATCTTTG CAGCTTTGCT ATTCTGTACC TCTGCCGCAA TC - #GTTGCTGG        2160                                                                           - AGATGCTTCG GACGCTGTGT GCGAGTTGGG CCTGCCACGC ATGTTTTGGG CT - #CCACCGGG        2220                                                                           - CAACGAGTTT CCAAACTGGC GCTCATTGAT TTGTGTGACC ACTTTTCAAA GC - #CCACCATC        2280                                                                           - GATGTTGTGG GCATGGCAAC TGGTTGGAGC GGATGTTACA CAGGAACCGC CG - #CAATGGAG        2340                                                                           - CGTCAGTGTG CCTCTACGGT GGACCCTCAC TCGTTCGACC AGAAGAAGGC AG - #GAGCGACT        2400                                                                           - GTTTACCTCA CCCCCCCTGT CAACAGCGGG TCAGCGCTGC AGTGCCTCAA TG - #TCATGTGG        2460                                                                           - AAGCGACCAA TTGGGTCCAC TGTCCTTGGG GAACAAACAG GAGCTGTTGT GA - #CGGCGGTC        2520                                                                           - AAGAGTATCT CTTTCTCACC TCCCTGCTGC GTCTCTACCA CTTTGCCCAC CC - #GACCCGGT        2580                                                                           - GTGACCGTTG TCGACCATGC TCTTTACAAC CGGTTGACTG CTTCAGGGGT CG - #ATCCCGCT        2640                                                                           - TTATTGCGTG TTGGGCAAGG TGATTTTCTA AAACTTAATC CGGGGTTCCG GC - #TGATAGGT        2700                                                                           - GGATGGATTT ATGGGATATG CTATTTTGTG TTGGTGGTTG TGTCAACTTT TA - #CCTGCTTA        2760                                                                           - CCTATCAAAT GTGGCATTGG CACCCGCGAC CCTTTCTGCC GCAGAGTGTT TT - #CTGTACCC        2820                                                                           - GTCACCAAGA CCCAAGAGCA CTGCCATGCT GGAATGTGTG CTAGCGCTGA AG - #GCATCTCT        2880                                                                           - CTGGACTCTC TGGGGTTAAC TCAGTTACAA AGTTACTGGA TCGCAGCCGT CA - #CTAGCGGA        2940                                                                           - TTAGTGATCT TGTTGGTCTG CCACCGCCTG GCCATCAGCG CCTTGGACTT GT - #TGACTCTA        3000                                                                           - GCTTCCCCTT TAGTGTTGCT TGTGTTCCCT TGGGCATCTG TGGGGCTTTT AC - #TTGCTTGC        3060                                                                           - AGTCTCGCTG GTGCTGCTGT GAAAATACAG TTGTTGGCGA CGCTTTTTGT GA - #ATCTGTTC        3120                                                                           - TTTCCCCAAG CTACCCTTGT CACTATGGGA TACTGGGCGT GCGTGGCGGC TT - #TGGCCGTT        3180                                                                           - TACAGTTTGA TGGGCTTGCG AGTGAAAGTG AATGTGCCCA TGTGTGTGAC AC - #CTGCCCAT        3240                                                                           - TTTCTGCTGC TGGCGAGGTC AGCTGGACAG TCAAGAGAGC AGATGCTCCG GG - #TCAGCGCT        3300                                                                           - GCTGCCCCCA CCAATTCACT GCTTGGAGTG GCTCGTGATT GTTATGTCAC AG - #GCACAACT        3360                                                                           - CGGCTGTACA TACCCAAGGA AGGCGGGATG GTGTTTGAAG GGCTATTCAG GT - #CACCGAAG        3420                                                                           - GCGCGCGGCA ACGTCGGCTT CGTGGCTGGT AGCAGCTACG GCACAGGGTC AG - #TGTGGACC        3480                                                                           - AGGAACAACG AGGTCGTCGT ACTGACAGCG TCACACGTGG TTGGCCGCGC TA - #ACATGGCC        3540                                                                           - ACTCTGAAGA TCGGTGACGC AATGCTGACT CTGACTTTCA AAAAGAATGG CG - #ACTTCGCC        3600                                                                           - GAGGCAGTGA CGACACAGTC CGAGCTCCCA GGCAATTGGC CACAGTTGCA TT - #TCGCCCAA        3660                                                                           - CCAACAACCG GGCCCGCTTC ATGGTGCACT GCCACAGGAG ATGAAGAAGG CT - #TGCTCAGT        3720                                                                           - GGCGAGGTTT GTCTGGCGTG GACTACTAGT GGCGACTCTG GATCTGCAGT GG - #TTCAGGGT        3780                                                                           - GACGCTGTGG TAGGGGTCCA CACCGGTTCG AACACAAGTG GTGTTGCCTA CG - #TGACCACC        3840                                                                           - CCAAGCGGAA AACTCCTTGG CGCCGACACC GTGACTTTGT CATCACTGTC AA - #AGCATTTC        3900                                                                           - ACAGGCCCTT TGACATCAAT CCCGAAGGAC ATCCCTGACA ACATTATTGC CG - #ATGTTGAT        3960                                                                           - GCTGTTCCTC GTTCTCTGGC CATGCTGATT GATGGCTTAT CCAATAGAGA GA - #GCAGCCTT        4020                                                                           - TCTGGACCTC AGTTGTTGTT AATTGCTTGT TTTATGTGGT CTTATCTTAA CC - #AACCTGCT        4080                                                                           - TACTTGCCTT ATGTGCTGGG CTTCTTTGCC GCTAACTTCT TCCTGCCAAA AA - #GTGTTGGC        4140                                                                           - CGCCCTGTGG TCACTGGGCT TCTATGGTTG TGCTGCCTCT TCACACCGCT TT - #CCATGCGC        4200                                                                           - TTGTGCTTGT TCCATCTGGT CTGTGCTACC GTCACGGGAA ACGTGATATC TT - #TGTGGTTC        4260                                                                           - TACATCACTG CCGCTGGCAC GTCTTACCTT TCTGAGATGT GGTTCGGAGG CT - #ATCCCACC        4320                                                                           - ATGTTGTTTG TGCCACGGTT CCTAGTGTAC CAGTTCCCCG GCTGGGCTAT TG - #GCACAGTA        4380                                                                           - CTAGCGGTAT GCAGCATCAC CATGCTGGCT GCTGCCCTCG GTCACACCCT GT - #TACTGGAT        4440                                                                           - GTGTTCTCCG CCTCAGGTCG CTTTGACAGG ACTTTCATGA TGAAATACTT CC - #TGGAGGGA        4500                                                                           - GGAGTGAAAG AGAGTGTCAC CGCCTCAGTC ACCCGCGCTT ATGGCAAACC AA - #TTACCCAG        4560                                                                           - GAGAGTCTCA CTGCAACATT AGCTGCCCTC ACTGATGATG ACTTCCAATT CC - #TCTCTGAT        4620                                                                           - GTGCTTGACT GTCGGGCCGT CCGATCGGCA ATGAATCTCG GTGCCGCTCT CA - #CAAGTTTT        4680                                                                           - CAAGTGGCGC AGTATCGTAA CATCCTTAAT GCATCCTTGC AAGTCGATCG TG - #ACGCTGCT        4740                                                                           - CGTAGTCGCA GACTAATGGC AAAACTGGCT GATTTTGCGG TTGAACAAGA AG - #TAACAGCT        4800                                                                           - GGAGACCGTG TTGTGGTTAT CGACGGTCTG GACCGCATGG CTCACTTCAA AG - #ACGATTTG        4860                                                                           - GTGCTGGTTC CTTTGACCAC CAAAGTAGTA GGCGGTTCTA GGTGCACCAT TT - #GTGACGTC        4920                                                                           - GTTAAGGAAG AAGCCAATGA CACCCCAGTT AAGCCAATGC CCAGCAGGAG AC - #GCCGCAAG        4980                                                                           - GGCCTGCCTA AAGGTGCTCA GTTGGAGTGG GACCGTCACC AGGAAGAGAA GA - #GGAACGCC        5040                                                                           - GGTGATGATG ATTTTGCGGT CTCGAATGAT TATGTCAAGA GAGTGCCAAA GT - #ACTGGGAT        5100                                                                           - CCCAGCGACA CCCGAGGCAC GACAGTGAAA ATCGCCGGCA CTACCTATCA GA - #AAGTGGTT        5160                                                                           - GACTATTCAG GCAATGTGCA TTACGTGGAG CATCAGGAAG ATCTGCTAGA CT - #ACGTGCTG        5220                                                                           - GGCAAGGGGA GCTATGAAGG CCTAGATCAG GACAAAGTGT TGGACCTCAC AA - #ACATGCTT        5280                                                                           - AAAGTGGACC CCACGGAGCT CTCCTCCAAA GACAAAGCCA AGGCGCGTCA CG - #TTGCTCAT        5340                                                                           - CTGCTGTTGG ATCTGGCTAA CCCAGTTGAG GCAGTGAATC AGTTAAACTG AG - #AGCGCCCC        5400                                                                           - ACATCTTTCC CGGCGATGTG GGGCGTCGGA CCTTTGCTGA CTCTAAAGAC AA - #GGGTTTCG        5460                                                                           - TGGCTCTACA CAGTCGCACA ATGTTTTTAG CTGCCCGGGA CTTTTTATTT AA - #CATCAAAT        5520                                                                           - TTGTGTGCGA CGAAGAGTTC ACAAAGACCC CAAAAGACAC ACTGCTTGGG TA - #CGTACGCG        5580                                                                           - CCTGCCCTGG TTACTGGTTT ATTTTCCGTC GTACGCACCG GTCGCTGATT GA - #TGCATACT        5640                                                                           - GGGACAGTAT GGAGTGCGTT TACGCGCTTC CCACCATATC TGATTTTGAT GT - #GAGCCCAG        5700                                                                           - GTGACGTCGC AGTGACGGGC GAGCGATGGG ATTTTGAATC TCCCGGAGGA GG - #CCGTGCAA        5760                                                                           - AACGTCTCAC AGCTGATCTG GTGCACGCTT TTCAAGGGTT CCACGGAGCC TC - #TTATTCCT        5820                                                                           - ATGATGACAA GGTGGCAGCT GCTGTCAGTG GTGACCCGTA TCGGTCGGAC GG - #CGTCTTGT        5880                                                                           - ATAACACCCG TTGGGGCAAC ATTCCATATT CTGTCCCAAC CAATGCTTTG GA - #AGCCACAG        5940                                                                           - CTTGCTACCG TGCTGGATGT GAGGCCGTTA CCGACGGGAC CAACGTCATC GC - #AACAATTG        6000                                                                           - GGCCCTTCCC GGAGCAACAA CCCATACCGG ACATCCCAAA GAGCGTGCTT GA - #CAACTGCG        6060                                                                           - CTGACATCAG CTGTGACGCT TTCATAGCGC CCGCTGCAGA GACAGCCCTG TG - #TGGAGATT        6120                                                                           - TAGAGAAATA CAACCTATCC ACGCAGGGTT TTGTGTTGCC TAGTGTTTTC TC - #CATGGTGC        6180                                                                           - GGGCGTACTT AAAAGAGGAG ATTGGAGACG CTCCACCACT CTACTTGCCA TC - #TACTGTAC        6240                                                                           - CATCTAAAAA TTCACAAGCC GGAATTAACG GCGCTGAGTT TCCTACAAAG TC - #TTTACAGA        6300                                                                           - GCTACTGTTT GATTGATGAC ATGGTGTCAC AGTCCATGAA AAGCAATCTA CA - #AACCGCCA        6360                                                                           - CCATGGCGAC TTGTAAACGG CAATACTGTT CCAAATACAA GATTAGGAGC AT - #TCTGGGCA        6420                                                                           - CCAACAATTA CATTGGCCTA GGTTTGCGTG CCTGCCTTTC GGGGGTTACG GC - #CGCATTCC        6480                                                                           - AAAAAGCTGG AAAGGATGGG TCACCGATTT ATTTGGGCAA GTCAAAATTC GA - #CCCGATAC        6540                                                                           - CAGCTCCTGA CAAGTACTGC CTTGAAACAG ACCTGGAGAG TTGTGATCGC TC - #CACCCCGG        6600                                                                           - CTTTGGTGCG TTGGTTCGCT ACTAATCTTA TTTTTGAGCT AGCTGGCCAG CC - #CGAGTTGG        6660                                                                           - TGCACAGCTA CGTGTTGAAT TGCTGTCACG ATCTAGTTGT GGCGGGTAGT GT - #AGCATTCA        6720                                                                           - CCAAACGCGG GGGTTTGTCA TCTGGAGACC CTATCACTTC CATTTCCAAT AC - #CATCTATT        6780                                                                           - CATTGGTGCT GTACACCCAG CACATGTTGC TATGTGGACT TGAAGGCTAT TT - #CCCAGAGA        6840                                                                           - TTGCAGAAAA ATATCTTGAT GGCAGCCTGG AGCTGCGGGA CATGTTCAAG TA - #CGTTCGAG        6900                                                                           - TGTACATCTA CTCGGACGAT GTGGTTCTAA CCACACCCAA CCAGCATTAC GC - #GGCCAGCT        6960                                                                           - TTGACCGCTG GGTCCCCCAC CTGCAGGCGC TGCTAGGTTT CAAGGTTGAC CC - #AAAGAAAA        7020                                                                           - CTGTGAACAC CAGCTCCCCT TCCTTTTTGG GCTGCCGGTT CAAGCAAGTG GA - #CGGCAAGT        7080                                                                           - GTTATCTAGC CAGTCTTCAG GACCGCGTTA CACGCTCTCT GTTATACCAC AT - #TGGTGCAA        7140                                                                           - AGAATCCCTC AGAGTACTAT GAAGCTGCTG TTTCCATCTT TAAGGACTCC AT - #TATCTGCT        7200                                                                           - GTGATGAAGA CTGGTGGACG GACCTCCATC GACGTATCAG TGGCGCTGCG CG - #TACCGACG        7260                                                                           - GAGTTGAGTT CCCCACCATT GAAATGTTAA CATCCTTCCG CACCAAGCAG TA - #TGAGAGTG        7320                                                                           - CCGTGTGCAC AGTTTGTGGG GCCGCCCCCG TGGCCAAGTC TGCTTGTGGA GG - #GTGGTTCT        7380                                                                           - GTGGCAATTG TGTCCCGTAC CACGCGGGTC ATTGTCACAC AACCTCGCTC TT - #CGCCAACT        7440                                                                           - GCGGGCACGA CATCATGTAC CGCTCCACTT ACTGCACAAT GTGTGAGGGT TC - #CCCAAAAC        7500                                                                           - AGATGGTACC AAAAGTGCCT CACCCGATCC TGGATCATTT GCTGTGCCAC AT - #TGATTACG        7560                                                                           - GCAGTAAAGA GGAACTAACT CTGGTAGTGG CGGATGGTCG AACAACATCA CC - #GCCCGGGC        7620                                                                           - GCTACAAAGT GGGTCACAAG GTAGTCGCCG TGGTTGCAGA TGTGGGAGGC AA - #CATTGTGT        7680                                                                           - TTGGGTGCGG TCCTGGATCA CACATCGCAG TACCACTTCA GGATACGCTC AA - #GGGCGTGG        7740                                                                           - TGGTGAATAA AGCTCTGAAG AACGCCGCCG CCTCTGAGTA CGTGGAAGGA CC - #CCCTGGGA        7800                                                                           - GTGGGAAGAC TTTTCACCTG GTCAAAGATG TGCTAGCCGT GGTCGGTAGC GC - #GACCTTGG        7860                                                                           - TTGTGCCCAC CCACGCGTCC ATGCTGGACT GCATCAACAA GCTCAAACAA GC - #GGGCGCCG        7920                                                                           - ATCCATACTT TGTGGTGCCC AAGTATACAG TTCTTGACTT TCCCCGGCCT GG - #CAGTGGAA        7980                                                                           - ACATCACAGT GCGACTGCCA CAGGTCGGAA CCAGTGAGGG AGAAACCTTT GT - #GGATGAGG        8040                                                                           - TGGCCTACTT CTCACCAGTG GATCTGGCGC GCATTTTAAC CCAGGGTCGA GT - #CAAGGGTT        8100                                                                           - ACGGTGATTT AAATCAGCTC GGGTGCGTCG GACCCGCGAG CGTGCCACGT AA - #CCTTTGGC        8160                                                                           - TCCGACATTT TGTCAGCCTG GAGCCCTTGC GAGTGTGCCA TCGATTCGGC GC - #TGCTGTGT        8220                                                                           - GTGATTTGAT CAAGGGCATT TATCCTTATT ATGAGCCAGC TCCACATACC AC - #TAAAGTGG        8280                                                                           - TGTTTGTGCC AAATCCAGAC TTTGAGAAAG GTGTAGTCAT CACCGCCTAC CA - #CAAAGATC        8340                                                                           - GCGGTCTTGG TCACCGCACA ATTGATTCAA TTCAAGGCTG TACATTCCCT GT - #TGTGACTC        8400                                                                           - TTCGACTGCC CACACCCCAA TCACTGACGC GCCCGCGCGC AGTTGTGGCG GT - #TACTAGGG        8460                                                                           - CGTCTCAGGA ATTATACATC TACGACCCCT TTGATCAGCT TAGCGGGTTG TT - #GAAGTTCA        8520                                                                           - CCAAGGAAGC AGAGGCGCAG GACTTGATCC ATGGCCCACC TACAGCATGC CA - #CCTGGGCC        8580                                                                           - AAGAAATTGA CCTTTGGTCC AATGAGGGCC TCGAATATTA CAAGGAAGTC AA - #CCTGCTGT        8640                                                                           - ACACACACGT CCCCATCAAG GATGGTGTAA TACACAGTTA CCCTAATTGT GG - #CCCTGCCT        8700                                                                           - GTGGCTGGGA AAAGCAATCC AACAAAATTT CGTGCCTCCC GAGAGTGGCA CA - #AAATTTGG        8760                                                                           - GCTACCACTA TTCCCCAGAC TTACCAGGAT TTTGCCCCAT ACCAAAAGAA CT - #CGCTGAGC        8820                                                                           - ATTGGCCCGT AGTGTCCAAT GATAGATACC CGAATTGCTT GCAAATTACC TT - #ACAGCAAG        8880                                                                           - TATGTGAACT CAGTAAACCG TGCTCAGCGG GCTATATGGT TGGACAATCT GT - #TTTCGTGC        8940                                                                           - AGACGCCTGG TGTGACATCT TACTGGCTTA CTGAATGGGT CGACGGCAAA GC - #GCGTGCTC        9000                                                                           - TACCAGATTC CTTATTCTCG TCCGGTAGGT TCGAGACTAA CAGCCGCGCT TT - #CCTCGATG        9060                                                                           - AAGCCGAGGA AAAGTTTGCC GCCGCTCACC CTCATGCCTG TTTGGGAGAA AT - #TAATAAGT        9120                                                                           - CCACCGTGGG AGGATCCCAC TTCATCTTTT CCCAATATTT ACCACCATTG CT - #ACCCGCAG        9180                                                                           - ACGCTGTTGC CCTGGTAGGT GCTTCATTGG CTGGGAAAGC TGCTAAAGCT GC - #TTGCAGCG        9240                                                                           - TTGTTGATGT CTATGCTCCA TCATTTGAAC CTTATCTACA CCCTGAGACA CT - #GAGTCGCG        9300                                                                           - TGTACAAGAT TATGATCGAT TTCAAGCCGT GTAGGCTTAT GGTGTGGAGA AA - #CGCGACCT        9360                                                                           - TTTATGTCCA AGAGGGTGTT GATGCAGTTA CATCAGCACT AGCAGCTGTG TC - #CAAACTCA        9420                                                                           - TCAAAGTGCC GGCCAATGAG CCTGTTTCAT TCCATGTGGC ATCAGGGTAC AG - #AACCAACG        9480                                                                           - CGCTGGTAGC GCCCCAGGCT AAAATTTCAA TTGGAGCCTA CGCCGCCGAG TG - #GGCACTGT        9540                                                                           - CAACTGAACC GCCACCTGCT GGTTATGCGA TCGTGCGGCG ATATATTGTA AA - #GAGGCTCC        9600                                                                           - TCAGCTCAAC AGAAGTGTTC TTGTGCCGCA GGGGTGTTGT GTCTTCCACC TC - #AGTGCAGA        9660                                                                           - CCATTTGTGC ACTAGAGGGA TGTAAACCTC TGTTCAACTT CTTACAAATT GG - #TTCAGTCA        9720                                                                           - TTGGGCCCGT GTGATGGGCT TAGTGTGGTC ACTGATTTCA AATTCTATTC AG - #ACTATTAT        9780                                                                           - TGCTGATTTT GCTATTTCTG TGATTGATGC AGCGCTTTTC TTTCTCATGC TA - #CTTGCATT        9840                                                                           - GGCTGTTGTT ACTGTGTTTC TTTTCTGGCT CATTGTTGCC ATCGGCCGCA GC - #TTGGTGGC        9900                                                                           - GCGGTGTTCA CGAGGTGCGC GTTACAGACC TGTTTAAGGA TTTGCAGTGC GA - #CAACCTGC        9960                                                                           - GCGCGAAAGA TGCCTTCCCG AGTCTGGGAT ATGCTCTGTC GATTGGCCAG TC - #GAGGCTAT        10020                                                                          - CGTATATGCT GCAGGATTGG TTGCTTGCTG CGCACCGCAA GGAAGTTATG CC - #TTCCAATA        10080                                                                          - TCATGCCTAT GCCCGGTCTT ACTCCTGATT GCTTTGACCA TCTGGAGTCT TC - #TAGCTATG        10140                                                                          - CTCCATTTAT CAATGCCTAT CGGCAGGCAA TTTTGAGTCA ATACCCACAA GA - #GCTCCAGC        10200                                                                          - TCGAAGCCAT CAACTGTAAA TTGCTTGCTG TGGTTGCACC GGCATTGTAT CA - #TAATTACC        10260                                                                          - ATCTAGCCAA TTTGACCGGA CCGGCCACAT GGGTCGTGCC TACAGTGGGC CA - #GTTGCACT        10320                                                                          - ATTATGCTTC TTCCTCTATT TTTGCTTCAT CTGTGGAAGT GTTGGCAGCA AT - #AATACTAC        10380                                                                          - TATTTGCATG CATACCACTA GTGACACGAG TGTACATCTC TTTTACGCGG CT - #AATGTCAC        10440                                                                          - CTTCCCGTCG CACTTCCAGC GGCACTTTGC CGCGGCGCAA GATTTTGTAG TG - #CACACGGG        10500                                                                          - TTATGAATAT GCCGGGGTCA CTATGTTAGT GCACTTGTTT GCCAACTTGG TT - #CTGACATT        10560                                                                          - TCCGAGCTTA GTTAATTGTT CCCGCCCTGT GAATGTCTTT GCTAATGCTT CT - #TGCGTGCA        10620                                                                          - AGTGGTTTGT AGTCATACCA ACTCAACTAC TGGCTTGGGT CAACTTTCTT TT - #TCCTTTGT        10680                                                                          - AGATGAAGAT CTACGGCTGC ATATCAGGCC TACTCTTATT TGTTGGTTTG CC - #TTGTTGTT        10740                                                                          - GGTGCACTTT CTACCCATGC CACGCTGCAG AGGCTCGTAA TTTTACTTAC AT - #TAGTCATG        10800                                                                          - GATTGGGCCA CGTGCACGGT CATGAGGGGT GTAGGAATTT TATTAATGTC AC - #TCATTCTG        10860                                                                          - CATTTCTTTA TCTTAATCCC ACCACTCCCA CTGCGCCGGC TATAACTCAT TG - #TTTACTTC        10920                                                                          - TGGTTCTGGC AGCCAAAATG GAACACCCAA ACGCTACTAT CTGGCTGCAG CT - #GCAGCCGT        10980                                                                          - TTGGGTATCA TGTGGCTGGC GATGTCATTG TCAACTTGGA AGAGGACAAG AG - #GCATCCTT        11040                                                                          - ACTTTAAACT TTTGAGAGCG CCGGCTTTAC CGCTTGGTTT TGTGGCTATA GT - #TTATGTTC        11100                                                                          - TTTTACGACT GGTACGTTGG GCTCAACGAT GTTATCTATG ATTGTATTGC TA - #TTCTTGCT        11160                                                                          - TTGGGGTGCG CCATCACATG CTTACTTCTC ATACTACACC GCTCAGCGCT TC - #ACAGACTT        11220                                                                          - CACCTTGTGT ATGCTGACGG ATCGCGGCGT TATTGCCAAT TTGCTGCGAT AT - #GATGAGCA        11280                                                                          - CACTGCTTTG TACAATTGTT CCGCCAGTAA AACCTGTTGG TATTGCACAT TC - #CTGGACGA        11340                                                                          - ACAGATTATC ACGTTTGGAA CCGATTGTGA TGACACCTAC GCGGTCCCAG TT - #GCTGAGGT        11400                                                                          - CCTGGAACAG GCGCATGGAC CGTACAGTGC GCTGTTTGAT GACATGCCCC CT - #TTTATTTA        11460                                                                          - CTATGGCCGT GAATTCGGCA TAGTTGTGTT GGATGTGTTT ATGTTCTATC CC - #GTTTTAGT        11520                                                                          - TCTGTTTTTC TTATCAGTAC TACCCTATGC TACGCTTATT CTTGAAATGT GT - #GTATCTAT        11580                                                                          - TCTGTTTATA ATCTATGGCA TTTACAGCGG GGCCTACTTG GCCATGGGCA TA - #TTTGCGGC        11640                                                                          - CACGCTTGCT ATACATTCAA TTGTGGTCCT CCGCCAATTA CTGTGGTTAT GC - #CTGGCTTG        11700                                                                          - GCGATACCGC TGTACGCTTC ACGCGTCCTT TATATCAGCT GAGGGGAAAG TG - #TACCCCGT        11760                                                                          - AGACCCCGGA CTCCCGGTTG CCGCCGTGGG CAATCGGTTG TTAGTCCCAG GT - #AGGCCCAC        11820                                                                          - TATCGATTAT GCAGTGGCCT ACGGCAGCAA AGTCAACCTT GTGAGGTTGG GG - #GCAGCTGA        11880                                                                          - GGTATGGGAG CCATAGATTC ATTTTGTGGT GACGGGATTT TAGGTGAGTA TC - #TAGATTAC        11940                                                                          - TTTATTCTGT CCGTCCCACT CTTGCTGTTG CTTACTAGGT ATGTAGCATC TG - #GGTTAGTG        12000                                                                          - TATGTTTTGA CTGCCTTGTT CTATTCCTTT GTATTAGCAG CTTATATTTG GT - #TTGTTATA        12060                                                                          - GTTGGAAGAG CCTTTTCTAC TGCTTATGCT TTTGTGCTTT TGGCTGCTTT TC - #TGTTATTA        12120                                                                          - GTAATGAGGA TGATTGTGGG TATGATGCCT CGTCTTCGGT CCATTTTCAA CC - #ATCGCCAA        12180                                                                          - CTGGTGGTAG CTGATTTTGT GGACACACCT AGTGGACCTG TTCCCATCCC CC - #GCTCAACT        12240                                                                          - ACTCAGGTAG TGGTTCGCGG CAACGGGTAC ACCGCAGTTG GTAACAAGCT TG - #TCGATGGC        12300                                                                          - GTCAAGACGA TCACGTCCGC AGGCCGCCTC TTTTCGAAAC GGACGGCGGC GA - #CAGCCTAC        12360                                                                          - AAGCTACAAT GACCTACTGC GCATGTTTGG TCAGATGCGG GTCCGCAAAC CG - #CCCGCGCA        12420                                                                          - ACCCACTCAG GCTATTATTG CAGAGCCTGG AGACCTTAGG CATGATTTAA AT - #CAACAGGA        12480                                                                          - GCGCGCCACC CTTTCGTCGA ACGTACAACG GTTCTTCATG ATTGGGCATG GT - #TCACTCAC        12540                                                                          - TGCAGATGCC GGAGGACTCA CGTACACCGT CAGTTGGGTT CCTACCAAAC AA - #ATCCAGCG        12600                                                                          - CAAAGTTGCG CCTCCAGCAG GGCCGTAAGA CGTGGATATT CTCCTGTGTG GC - #GTCATGTT        12660                                                                          #          12687   ACCC AGGAACC                                                - (2) INFORMATION FOR SEQ ID NO: 2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 110 amino                                                          (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              #ID NO: 2:(xi) SEQUENCE DESCRIPTION: SEQ                                       -      Met Ala Ser Arg Arg Ser Arg Pro - # Gln Ala Ala Ser Phe Arg Asn         Gly                                                                            #   15                                                                         -      Arg Arg Arg Gln Pro Thr Ser Tyr - # Asn Asp Leu Leu Arg Met Phe         Gly                                                                            #                 30                                                           -      Gln Met Arg Val Arg Lys Pro Pro - # Ala Gln Pro Thr Gln Ala Ile         Ile                                                                            #             45                                                               -      Ala Glu Pro Gly Asp Leu Arg His - # Asp Leu Asn Gln Gln Glu Arg         Ala                                                                            #         60                                                                   -      Thr Leu Ser Ser Asn Val Gln Arg - # Phe Phe Met Ile Gly His Gly         Ser                                                                            #     80                                                                       -      Leu Thr Ala Asp Ala Gly Gly Leu - # Thr Tyr Thr Val Ser Trp Val         Pro                                                                            #   95                                                                         -      Thr Lys Gln Ile Gln Arg Lys Val - # Ala Pro Pro Ala Gly Pro             #                110                                                           - (2) INFORMATION FOR SEQ ID NO: 3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 69 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              #ID NO: 3:(xi) SEQUENCE DESCRIPTION: SEQ                                       -      Met Ala Ser Arg Arg Ser Arg Pro - # Gln Ala Ala Ser Phe Arg Asn         Gly                                                                            #   15                                                                         -      Arg Arg Arg Gln Pro Thr Ser Tyr - # Asn Asp Leu Leu Arg Met Phe         Gly                                                                            #                 30                                                           -      Gln Met Arg Val Arg Lys Pro Pro - # Ala Gln Pro Thr Gln Ala Ile         Ile                                                                            #             45                                                               -      Ala Glu Pro Gly Asp Leu Arg His - # Asp Leu Asn Gln Gln Glu Arg         Ala                                                                            #         60                                                                   -      Thr Leu Ser Ser Asn                                                          65                                                                        - (2) INFORMATION FOR SEQ ID NO: 4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                           #acids    (A) LENGTH: 28 amino                                                           (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                 -     (ii) MOLECULE TYPE: peptide                                              #ID NO: 4:(xi) SEQUENCE DESCRIPTION: SEQ                                       -      Met Ala Ser Arg Arg Ser Arg Pro - # Gln Ala Ala Ser Phe Arg Asn         Gly                                                                            #   15                                                                         -      Arg Arg Arg Gln Pro Thr Ser Tyr - # Asn Asp Leu Leu                     #                 25                                                           __________________________________________________________________________ 

What is claimed is:
 1. An antigenic peptide fragment of equine arteritis virus (EAV) viral nucleocapsid (N) (SEQ ID NO: 2) which fragment is selected from the group consisting of:a) amino acid residues 1 to 69 of EAV N (SEQ ID NO: 3); b) amino acid residues 1 to 28 of EAV N (SEQ ID NO: 4); and c) a sequence of amino acid residues having at least 90% homology to any one of sequences (a) or (b).
 2. The peptide fragment according to claim 1 wherein the peptide fragment is attached to a label and wherein the label is selected from the group consisting of: a radiolabel; a chemical label; an enzyme; and a cofactor.
 3. A test kit for testing for the presence of antibodies to equine arteritis virus (EAV) which kit comprises a peptide fragment according to claim
 2. 4. A method for testing for the presence of antibodies to equine arteritis virus (EAV) which comprises the steps of selecting a peptide fragment as defined in claim 1 as a specific binding agent, incubating a sample to be screened for EAV antibodies in contact with the specific binding agent, and identifying any EAV antibodies present.
 5. The method according to claim 4 wherein the EAV antibodies are identified by ELISA, or the specific binding agent is an immobilized binding agent or labeled secondary binding agent.
 6. The method according to claim 4 wherein the peptide fragment is labeled and wherein the label is selected from the group consisting of: a radiolabel; a chemical label; an enzyme; and a cofactor.
 7. A test kit for testing for the presence of antibodies to equine arteritis virus (EAV) which kit comprises a peptide fragment as defined in claim
 1. 8. A composition which comprises as an active ingredient an antigenic peptide fragment according to claim 1, wherein the peptide fragment is conjugated to glutathione-s-transferase (GST) to form a peptide conjugate.
 9. The peptide conjugate according to claim 8 wherein the peptide fragment is attached to a label and wherein the label is selected from the group consisting of: a radiolabel; a chemical label; an enzyme; and a cofactor.
 10. A test kit for testing for the presence of antibodies to equine arteritis virus (EAV) which kit comprises a peptide conjugate according to claim
 9. 11. A method for testing for the presence of antibodies to equine arteritis virus (EAV) which comprises the steps of selecting a peptide conjugate as defined in claim 8 as a specific binding agent, incubating a sample to be screened for EAV antibodies in contact with the specific binding agent, and identifying any EAV antibodies present.
 12. The method according to claim 11 wherein the EAV antibodies are identified by ELISA, or the specific binding agent is an immobilized binding agent or labeled secondary binding agent.
 13. The method according to claim 11 wherein the peptide conjugate is labeled and wherein the label is selected from the group consisting of: a radiolabel; a chemical label; an enzyme; and a cofactor.
 14. A test kit for testing for the presence of antibodies to equine arteritis virus (EAV) which kit comprises a peptide conjugate according to claim
 2. 